CN113699150A - 一种敲减PKR的Marc-145细胞系 - Google Patents

一种敲减PKR的Marc-145细胞系 Download PDF

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CN113699150A
CN113699150A CN202110966613.0A CN202110966613A CN113699150A CN 113699150 A CN113699150 A CN 113699150A CN 202110966613 A CN202110966613 A CN 202110966613A CN 113699150 A CN113699150 A CN 113699150A
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肖跃强
杨慧
王文秀
魏凤
唐娜
李峰
沈志强
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Shandong Binzhou Animal Science & Veterinary Medicine Academy
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Abstract

本发明属于分子生物学和细胞生物学领域,具体涉及一种PKR基因敲减Marc‑145细胞系。本发明提供了一种利用RNAi敲减PKR基因的siRNA的正向和反向序列如SEQ ID NO:1‑2所示;shRNA的序列如SEQ ID NO:3所示。将含如SEQ ID NO:3所示目的基因序列的表达载体转染Marc‑145细胞系,筛选后可获得能够稳定遗传的PKR蛋白表达量降低的细胞系:Marc‑145∧143,该细胞系中PKR蛋白表达量降低不低于90%。该siRNA、shRNA或Marc‑145∧143细胞系可用于生产预防或治疗NDV、PRRSV或PRV的药剂或疫苗。

Description

一种敲减PKR的Marc-145细胞系
技术领域
本发明属于分子生物学和细胞生物学领域,具体涉及一种PKR基因敲减Marc-145细胞系。
背景技术
干扰素诱导的双链RNA激活的蛋白激酶(interferon-induced, double-stranded(ds)RNA-activated kinase,PKR)是一种丝氨酸-苏氨酸激酶,它能够被细胞、病毒的双链RNA(dsRNA)或合成类似物(例如polyI:C)激活,在宿主抗病毒防御过程中发挥重要作用。其抗病毒活性主要通过单体PKR与病毒复制过程中产生的双链RNA(dsRNA)结合,形成二聚体并发生自身磷酸化,之后催化其底物-真核生物翻译起始因子2α(alpha subunit of theeukaryotic initiation factore,eIF2α)磷酸化,导致病毒蛋白的合成受到抑制,从而产生抗病毒作用。
有研究表明[1],以siRNA转染下调Hela细胞PKR的表达能够促进鸡新城疫病毒(Newcastle disease virus, NDV)的增殖,转染重组质粒上调PKR的表达,NDV的增殖受到明显的抑制。
Xiao Y等[2]研究揭示,PRRSV在感染猪肺泡巨噬细胞(porcine alveolarmacrophage,PAM)早期通过抑制PKR活性从而促进自身的增殖。Marc-145细胞是支持猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)体外复制增殖的重要细胞系,克隆自MA-104细胞系,是源于长尾黑颚猴(Cercopithecus aethiops)的一种肾细胞系。Wang X等[3]研究PRRSV在Macr-145细胞感染研究时,病毒感染12~24 h能够短暂的活化PKR,并且在病毒感染前,细胞转染PKR基因小干扰RNA(smallinterfering RNA,siRNA),与对照相比,PRRSV基因转录、蛋白合成、增殖滴度均降低,说明PKR促进了PRRSV在Marc-145细胞的增殖,但没有进行细胞系构建并开展相关研究。可见,PRRSV在天然宿主细胞PAM及体外培养初代细胞Marc-145增殖过程中,PKR发挥了不尽相同的作用。
猪伪狂犬病病毒(Pseudorabies virus,PRV)感染宿主细胞范围比较广,有研究揭示[4],当PRV感染MDBK(牛肾细胞)时,PKR与PKR样内质网激酶(PKR-like ER kinase,PERK)的表达均下降,以PKR抑制剂2-氨基嘌呤处理病毒感染的细胞,蛋白表达能够部分恢复; XuS等[5]研究揭示,PRV感染PK-15细胞时,在感染早期PKR被活化,感染晚期其活性受到PRV蛋白的抑制,并且在感染早期,PRV感染Vero细胞时能够抑制eIF2α的磷酸化,然后促进自身的增殖,而eIF2α的磷酸化是由PRV感染后活化的PERK介导的。可见,PKR理论上对PRV的增殖有可能产生抑制作用。
参考文献:
[1] Zhang S, Sun Y, Chen H, Dai Y, Zhan Y, Yu S, Qiu X, Tan L, SongC, Ding C. Activation of the PKR/eIF2α signaling cascade inhibits replicationof Newcastle disease virus. Virol J. 2014 Mar 31;11:62.
[2] Xiao Y, Ma Z, Wang R, Yang L, Nan Y, Zhang YJ. Downregulation ofprotein kinase PKR activation by porcine reproductive and respiratorysyndrome virus at its early stage infection. Vet Microbiol. 2016 May 1;187:1-7.
[3] Wang X, Zhang H, Abel AM, Nelson E. Protein kinase R (PKR) playsa pro-viral role in porcine reproductive and respiratory syndrome virus(PRRSV) replication by modulating viral gene transcription. Arch Virol. 2016Feb;161(2):327-33.
[4] Wong ML, Yen YR. Protein synthesis in pseudorabies virus-infectedcells: decreased expression of protein kinase PKR, and effects of 2-aminopurine and adenine. Virus Res. 1998 Aug;56(2):199-206.
[5] Xu S, Chen D, Chen D, Hu Q, Zhou L, Ge X, Han J, Guo X, Yang H.Pseudorabies virus infection inhibits stress granules formation viadephosphorylating eIF2α. Vet Microbiol. 2020 Aug;247:108786。
发明内容
针对现有技术中的问题,本发明提供一种PKR基因敲减的Marc-145细胞系,能够促进新城疫病毒(NDV)增殖的同时抑制猪繁殖与呼吸综合征病毒(PRRSV)和猪伪狂犬病毒(PRV)增殖。
为实现上述目的,本发明采用如下技术方案。
一种敲减PKR基因的siRNA,其正向和反向核苷酸序列如SEQ ID NO:1-2所示。
一种敲减PKR基因的shRNA,其模板序列的核苷酸序列如SEQ ID NO:3所示。
一种包含如SEQ ID NO:1-2或SEQ ID NO:3所示目的基因序列的表达载体。所述表达载体是病毒、真菌或细菌表达载体。
一种包含上述表达载体的细胞。优选地,所述细胞选自家禽或哺乳动物细胞系,如,鸡胚细胞、HEK293细胞、Marc-145细胞或Vero细胞。
一种敲减PKR的Marc-145细胞系的构建方法,包括以下步骤:将含如SEQ ID NO:3所示目的基因序列的表达载体转染Marc-145细胞系,筛选获得能够稳定遗传的PKR蛋白表达量降低的细胞,即得。
一种上述方法获得的敲减PKR的Marc-145细胞系,PKR蛋白表达量降低不低于90%。
上述敲减PKR基因的siRNA、shRNA,上述包含如SEQ ID NO:1-2或SEQ ID NO:3所示目的基因序列的表达载体及其细胞,上述敲减PKR的Marc-145细胞系可应用于生产预防和治疗新城疫病毒(NDV)、猪繁殖与呼吸综合征病毒(PRRSV)或猪伪狂犬病毒(PRV)的药物。
本发明具有以下优点:
本发明提供的siRNA和shRNA可以显著降低Marc-145细胞PKR基因的表达量。通过该shRNA获得的敲减PKR的Marc-145细胞系可以稳定传代,PKR表达可长期被抑制。该稳传细胞系Marc-145∧143能够显著抑制PRRSV与PRV的增殖,且能够促进NDV的增殖,可用于治疗性药剂、疫苗等生物制品的生产和病毒宿主互作研究。
附图说明
图1为Marc-145细胞PKR基因扩增产物1%琼脂糖电泳图;
图2为pC1-PKR的荧光显微图片,其中,A为pEGFP-C1表达对照(100×),B为pC1-PKR转染表达(100×);
图3为pC1-PKR表达EGFP的Western Blot;其中,M为PageRulerTM预染蛋白Marker;1为HEK293细胞对照,2~3为pC1-PKR表达检测,4为pEGFP-C1表达检测;
图4为不同siRNA转染后HEK293细胞表达EGFP的Western Blot;其中,M为预染蛋白Marker,1为未转染,2为PKR1296;3为PKR143,4为PKR1058,5为PKR295,6为PKR1870S1C1,7为NC;
图5为shRNA稳定表达Marc-145细胞系(200×);其中,A为Marc-145∧143细胞系,B为Marc-145∧295细胞系,C为Marc-145∧1870S1C1细胞系,D为Marc-145∧NC细胞系,E为Marc-145细胞;1为潮霉素B筛选存活细胞,2为筛选后的细胞系或者正常Marc-145细胞;
图6为shRNA稳定表达细胞系PKR下调表达与XZ06a-EGFP感染后病毒M蛋白、重组EGFP的表达。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 siRNA和shRNA的设计和筛选
一、Marc-145的PKR基因克隆
根据已公开的Marc-145近源种PKR基因(GeneBank登录号:XM_015112081)设计引物,具体序列见表1。提取Marc-145细胞的总RNA,反转录获得cDNA,采用高保真酶Q5进行目的基因扩增。
表1 Marc-145细胞PKR基因扩增引物
Figure 339716DEST_PATH_IMAGE001
PCR产物以1%琼脂糖电泳(图1),回收大小符合目的基因(1653 bp)的产物,在3’端加碱基“A”,克隆至pMD18-T载体并测序,序列如SEQ ID NO:7所示。将测序正确的质粒保存备用。
二、PKR融合EGFP表达载体构建
根据表2中的序列分别合成引物PKR+C1-F和PKR+C1-FR,对Marc-145的PKR基因进行PCR扩增,PCR产物回收后以Sal I+BamH I双酶切,与相应酶切的pEGFP-C1质粒分别连接转化,筛选阳性克隆并测序,验证正确的质粒命名为pC1-PKR。
表2 PKR、EGFP融合蛋白载体构建引物
Figure 69775DEST_PATH_IMAGE002
将构建正确的质粒用Lipofectamine® 2000转染HEK293细胞,剂量为1 µg/孔,具体方法根据说明书进行。通过荧光显微镜观察(图2)、Western Blot(图3)检测EGFP蛋白表达,表明pC1-PKR成功转染HEK293细胞。
三、siRNA的筛选
根据Marc-145细胞PKR序列选择靶标序列并设计表3中siRNA PKR143、PKR295、PKR1058、PKR1296,同时设置无关siRNA为阴性对照(NC)和阳性对照PKR1870S1C1(Sigma-Aldrich,TRCN0000196400)。将EGFP融合表达质粒pC1-PKR(剂量0.5 µg)与PRK siRNA(剂量0.5 µg)共转染HEK 293细胞。Western Blot检测EGFP-PKR融合蛋白表达量(图4)。结果显示,PKR143、PKR295、PKR1870S1C1均能够下调PKR的表达,以PKR-1870S1C1下调效率最高,因此,选择143、295和1870S1C1的靶标序列设计shRNA进一步构建稳定表达细胞系。
表3 PKR基因的siRNA序列
Figure 535392DEST_PATH_IMAGE003
实施例2 shRNA稳定表达Marc-145细胞系的构建
按照表4合成包含BamH I和Hind III酶切位点的用于产生shRNA的DNA序列,同时设置无关shRNA模板为阴性对照(NC)和阳性对照PKR 1870S1C1(Sigma-Aldrich,TRCN0000196400)模板。将shRNA以BamH I+Hind III双酶切,用T4 DNA连接酶连接到同样双酶切的pSilencer™ 2.1-U6 hygro载体,转化Amp抗性筛选,挑取单菌落过夜培养后提取质粒,测序验证正确的分别命名为pShRNA-PKR NC、pShRNA-PKR 143、pShRNA-PKR 295和pShRNA-PKR 1870S1C1。
表4 Marc-145细胞PKR基因shRNA的DNA序列
Figure 263176DEST_PATH_IMAGE004
选择上述构建的pShRNA-PKR转染Marc-145,以300 µg/mL潮霉素B筛选,7 d后大部分细胞死亡,但随着时间延长,局部出现单个细胞克隆生长,待细胞克隆形成细胞簇后消化传代,并进一步克隆纯化,并持续抗性筛选培养,最后获得shRNA稳定表达的细胞系并分别命名为为Marc-145∧143、Marc-145∧295、Marc-145∧1870S1C1(阳性对照细胞系)、Marc-145∧NC(阴性对照细胞系),记为F0代,构建的细胞系显微图像如图5,箭头指示了转染shRNA后潮霉素B筛选下的存活细胞的最初形态。Western blot检测各细胞系PKR的下调表达,如图6所示:Marc-145∧143细胞系PKR的表达大幅度,下调比例在90%以上,Marc-145∧295、Marc-145∧1870S1C1细胞系PKR表达下调不显著,shRNA载体对照细胞系Marc-145∧NC细胞系的PKR的表达未见显著变化。
实施例3 Marc-145∧143对NDV、PRRSV和PRV增殖的影响
按照唐娜,等(唐娜, 杨慧, 刘磊,等. 表达EGFP重组猪繁殖与呼吸综合征病毒的构建与鉴定[J]. 中国动物传染病学报, 28(4):8.)的方法构建表达EGFP的PRRSV XZ06a-EGFP重组病毒。
一、PRRSV感染
将PRRSV XZ06a-EGFP分别感染Marc-145细胞、Marc-145∧143、Marc-145∧295、Marc-145∧1870S1C1及Marc-145∧NC的F3代细胞,感染剂量为MOI= 0.1,通过观察病毒致细胞病变效应(CPE),Western Blot检测PRRSV主要蛋白与EGFP表达,分析PKR下调表达对PRRSV增殖的影响。
结果显示,构建的细胞系感染0.1 MOI PRRSV XZ06a-EGFP,72 h~96 h后Marc-145∧143和Marc-145∧1870S1C1细胞均未出现明显的CPE,而Marc-145、对照细胞系Marc-145∧NC、Marc-145∧295细胞均呈现出明显的CPE;收获感染48 h后细胞,SDS-PAGE上样缓冲液煮沸5 min,蛋白电泳并转印PVDF膜,Western blot检测EGFP的表达,结果如图6所示,可见,重组病毒接种Marc-145、Marc-145∧NC、Marc-145∧295后EGFP获得高效表达,即PRRSV XZ06a-EGFP接种上述细胞后均正常增殖,而接种Marc-145∧143、Marc-145∧1870S1C1细胞后EGFP表达效率均大幅度降低,尤其是Marc-145∧143细胞,EGFP表达效率很低,PRRSV的M蛋白检测结果与EGFP一致。以上结果表明,虽然Marc-145∧1870S1C1细胞PKR表达效率未见明显下降,但CPE观察与EGFP检测一致,说明所构建的细胞系Marc-145∧143、Marc-145∧1870S1C1可有效的抑制病毒的增殖。
二、PRV感染
将构克隆纯化的细胞系F3代细胞分别感染PRV,感染剂量为MOI= 0.1,细胞病变后收获病毒,然后将病毒液10倍倍比稀释,接种Marc-145细胞,每个稀释度8个重复,持续观察细胞病变至第7天,统计对应稀释度出现病变的孔数,Reed-Muench法测定增殖病毒的半数组织培养感染剂量(TCID50),分析PKR下调表达对PRV增殖的影响。
通过测定各细胞系培养的PRV TCID50,发现Marc-145∧143能够显著抑制PRV的增殖,培养的病毒TCID50仅为10-5.38/0.1 mL,显著低于Marc-145细胞、Marc-145∧NC、Marc-145∧295、Marc-145∧1870S1C1培养的病毒,分别为10-6.5/0.1 mL、10-6.34/0.1 mL、10-6.32/0.1 mL、10-6.13/0.1 mL,说明PKR下调表达抑制PRV的增殖。
三、NDV感染
以新城疫病毒(NDV)Mukteswar毒株(Ⅰ系疫苗株)分别感染Marc-145细胞、Marc-145∧143、Marc-145∧295、Marc-145∧1870S1C1及Marc-145∧NC的F3代细胞,感染剂量为MOI= 1。同时,以各细胞系的细胞培养上清处理Vero细胞,接种同样剂量NDV,排除所构建细胞系shRNA转录后激活干扰素通路,产生抗病毒活性。
NDV接种构建的细胞系培养上清处理的Vero细胞,出现典型病变后收获测定HA效价,与未进行任何处理的Vero细胞相比,病毒HA效价无任何差异,均为4 log2,表明构建的细胞系细胞培养上清对NDV在Vero增殖无影响,说明细胞系未产生干扰素,从而干扰PRRSV的增殖。NDV感染Marc-145细胞、对照细胞系Marc-145∧NC、Marc-145∧143、Marc-145∧295、Marc-145∧1870S1C1,24 h后Marc-145、Marc-145∧NC、Marc-145∧295细胞全部出现细胞融合病变,Marc-145∧143与Marc-145∧1870S1C1未出现典型的细胞病变,48 h后全部病变,分别收集感染24 h细胞培养上清与细胞并冻融破碎细胞(培养基同体积无菌PBS),分别测定HA效价,NDV感染细胞上清液HA效价无差异,均为1 log2,感染的Marc-145∧143细胞破碎后HA效价为5 log2,其它4个细胞系,虽然Marc-145∧1870S1C1在病变时间上与Marc-145∧143类似,但细胞破碎后HA效价无差别,为3 log2,说明Marc-145∧143细胞系促进了NDV的增殖,佐证了该细胞系PKR基因表达受到显著抑制。
序列表
<110> 山东省滨州畜牧兽医研究院
<120> 一种敲减PKR的Marc-145细胞系
<130> 20210813
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA-PKR143-F
<400> 1
gguagaucaa agaaggaagt t 21
<210> 2
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<212> DNA
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<220>
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cuuccuucuu ugaucuacct t 21
<210> 3
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<220>
<223> shRNA-PKR143
<400> 3
ggatccggta gatcaaagaa ggaagttcaa gagacttcct tctttgatct accttttttg 60
gaaagctt 68
<210> 4
<211> 19
<212> DNA
<213> Cercopithecus aethiops
<400> 4
ggtagatcaa agaaggaag 19
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> PKR-F
<400> 5
acaatggctg gtcatcttgt acc 23
<210> 6
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> PKR-R
<400> 6
acactaacat gtatgtcgtt ttttctctg 29
<210> 7
<211> 1653
<212> DNA
<213> Cercopithecus aethiops
<400> 7
atggctggtg atcttgcacc aggtttctac atggaggaac ttaataaata ccgtcagaag 60
cagggagtaa catttagcta tcaagaactg cctaatacag gacctccaca tgataggagg 120
tttacatttc gagttgtaac agatagaaga gaatttccag aagctgaagg tagatcaaag 180
aaggaagcaa aaaatgctgc agccaaatta gctgttgata tacttaataa ggaaagcaag 240
gtagttagtc ctttttcatt gacaacaacc agttcttcgg aaggattatc cattgggaac 300
tacataggcg ttgtcaatag aattgcccag aagaaaagac taactgtaaa ttataaacaa 360
tgtacatcgg gggtgcatgg gccagaagga tttcattata aatgcaaaat tggagagaaa 420
gaatatagta ttggtacagg ttctactaaa caggaagcaa aacaattggc cgctaaactt 480
gcattgcttg agatatcaga agaaacctca gtgaaacctg actccatgtc ccctggttct 540
tttgctgcta cgtgtgactc ccaaagcaac cctttagtga acaactcact tgcttctgaa 600
tcatcatctg aaaatgacgt ctcagcagat acatcagaga taaattctaa cagtggcagt 660
ttaaacaagt cttcattgtt tacgaatggt ctcagaaata accaaaggaa ggcaaaaaga 720
tctttggcac ctacatttga ccctcctgac atgaaaggac caaagtatac tgaggatgca 780
aggtttggca cagattttga agaaataaaa ttcattagcg tgggtggatt tggccaagtt 840
ttcaaagcaa aacacagaat tgacggaaag acttacgtta ttaaacgtgt taaatataat 900
agcaagaagg cagagcgtga agtaaaagca ttggcagaac ttgatcatgt aaatattgtt 960
cactacaatg gctgttggga tggacttgat tatgatcctg agatcagtgc ttatgatcct 1020
gagagccctg attttgatcc tgagaacaaa aaaaatagtt taagatcaaa gactaagtgc 1080
cttttcatcc aaatggaatt ctgtgaaaaa gggacattgg aggaatggat tgaagataga 1140
aaaggcaaga aactagacaa agttttggct ttggaactct ttgaacaaat aacaaaaggg 1200
ttggattata tacattcaaa aaatttaatt catagagatc ttaagccaag taatatattc 1260
ttagtagata caaaacaaat aaagattgga gactttggac ttgcaacatc tctgaaaaat 1320
gatggaaagc gaacaaggaa tacgggaact ttgcgataca tgagcccaga acagatttct 1380
ttgcaagact atggaaagga agtggacctc tacgctttgg ggctaattct tgctgaactt 1440
cttcatgtat gtgacactgc ttccgaaaca tcaaagtttt tcaaagacct acgggatggc 1500
accatctcag atgtgtttga taaaagagaa aaaactcttc tagagaaatt actctcaaag 1560
aaacctgagg accgacctaa cacatctgaa atactaagca ccttgactac gtggaagaaa 1620
agcccagaga aaaaggaacg acatacatgt tag 1653
<210> 8
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> PKR+C1-F
<400> 8
acagtcgaca tggctggtca tcttgtacc 29
<210> 9
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> PKR+C1-R
<400> 9
acaggatccc gacatgtatg tcgttttttc tctg 34
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
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<400> 10
acuaccguug uuauaggugt t 21
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<212> DNA
<213> Artificial Sequence
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gccagaagga uuucauuaua tt 22
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<211> 21
<212> DNA
<213> Artificial Sequence
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<223> siRNA-PKR295-R
<400> 13
uauaaugaaa uccuucuggt t 21
<210> 14
<211> 21
<212> DNA
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<223> siRNA-PKR1058-F
<400> 14
gcaagaaacu agacaaagut t 21
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA-PKR1058-R
<400> 15
acuuugucua guuucuugct t 21
<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
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<400> 16
gcaagacuau ggaaaggaat t 21
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA-PKR1296-R
<400> 17
uuccuuucca uagucuugct t 21
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA-PKR1870S1C1-F
<400> 18
gcugaacuuc uucauguaug utt 23
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA-PKR1870S1C1-R
<400> 19
acauacauga agaaguucag ctt 23
<210> 20
<211> 20
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<223> PKR295
<400> 20
gccagaagga tttcattata 20
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<213> Artificial Sequence
<220>
<223> PKR1058
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gcaagaaact agacaaagt 19
<210> 22
<211> 19
<212> DNA
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gcaagactat ggaaaggaa 19
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PKR1870
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gctgaacttc ttcatgtatg t 21
<210> 24
<211> 68
<212> DNA
<213> Artificial Sequence
<220>
<223> shRNA-NC
<400> 24
ggatccacta ccgttgttat aggtgttcaa gagacaccta taacaacggt agtttttttg 60
gaaagctt 68
<210> 25
<211> 69
<212> DNA
<213> Artificial Sequence
<220>
<223> shRNA-PKR295
<400> 25
ggatccgcca gaaggatttc attatattca agagatataa tgaaatcctt ctggtttttt 60
ggaaagctt 69
<210> 26
<211> 72
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA-PKR1870S1C1
<400> 26
ggatccgctg aacttcttca tgtatgtttc aagagaacat acatgaagaa gttcagcttt 60
tttggaaagc tt 72

Claims (10)

1.一种敲减PKR基因的siRNA,其正向和反向核苷酸序列如SEQ ID NO:1-2所示。
2. 一种敲减PKR基因的shRNA,其模板序列的核苷酸序列如SEQ ID NO:3所示。
3. 一种包含如SEQ ID NO:1-2或SEQ ID NO:3所示目的基因序列的表达载体。
4.根据权利要求3所述的表达载体,其特征在于,表达载体选自病毒、真菌或细菌表达载体。
5.一种包含权利要求3或4所述表达载体的细胞。
6.根据权利要求5所述的细胞,其特征在于,所述细胞选自家禽或哺乳动物细胞系。
7.根据权利要求5所述的细胞,其特征在于,所述细胞选自鸡胚细胞、HEK293细胞、Marc-145细胞或Vero细胞。
8. 一种敲减PKR的Marc-145细胞系的构建方法,其特征在于,包括以下步骤:将含如SEQ ID NO:3所示目的基因序列的表达载体转染Marc-145细胞系,筛选获得能够稳定遗传的PKR蛋白表达量降低的细胞。
9.一种权利要求8所述的方法获得的敲减PKR的Marc-145细胞系,其特征在于,PKR蛋白表达量降低不低于90%。
10.一种如权利要求1所述的敲减PKR基因的siRNA,如权利要求2所述的敲减PKR基因的shRNA,如权利要求4所述的表达载体,如权利要求5-7任一所述的细胞或如权利要求9所述的敲减PKR的Marc-145细胞系在生产预防和治疗新城疫病毒、猪繁殖与呼吸综合征病毒或猪伪狂犬病毒药物中的应用。
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